Enantioselectivity of the musk odor sensation

This brief review, the summary of a talk at the Symposium on Biological Chirality 2000 in Szeged, Hungary, illustrates what chiral recognition tells us about the molecular parameters of the musk odor sensation. While the enantioselectivity of odor perception is strong evidence for the key role of proteinogenic receptors in the molecular mechanism of olfaction, the quantitative and qualitative odor differences of enantiomers are often not very pronounced, as in the case of muscone (17/26). In those cases, however, where there is strong enantiodiscrimination, we find most intense musk odorants with very low odor thresholds, such as (-)-(12R)-12-methyl-9-oxa-14-tetradecanolide (35), (12R;9Z)-12-methyl-14-tetradec-9-enolide [(R)-Nirvanolide, 38], and (-)-(4S;7R)-1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta[g]-2-benzopyran [(-)-(4S;7R)-Galaxolide, 57], the latter being rather rigid. We thus can assume the geometry of the musk receptor to be fairly complementary to these compounds, which therefore can serve as templates for the design of new musk odorants.     

From Cassyrane to Cashmeran – The Molecular Parameters of Odorants

This review, including some new experimental results, is the summary of a talk at the ‘flavors & fragrances 2013’ conference in Leipzig, organized jointly by the GDCh, the Liebig‐Vereinigung, and the EuCheMS. After times of searching for natural odor principles and serendipitous discoveries by chemical inspiration, directed odorant design today offers the highest hit rates for the discovery of new odorants, although serendipity still plays a role. Keeping intact the electronic shape required for a certain olfactophore‐binding geometry, one can add or subtract structural elements, rigidify molecular structures, or introduce more structural flexibility. To find out which structural features are critical, the ‘seco‐approach’, in which different fragments are removed by cutting strategic bonds, is the most analytical. Following this approach, such ingredients as Serenolide, Sylkolide, and Pomarose were designed. Transferring this design principle from the family of damascones to that of the theaspiranes led to the discovery of Cassyrane, though completely different structural features turned out to be relevant. Application of the seco‐concept to a 3,7a‐substituted 2,6,7,7a‐tetrahydro‐1H‐inden‐5‐yl musk lead structure derived from carotol resulted in the discovery of a new family of dienone musks with novel structure? odor correlations. However, cutting the C(2)? O bond of Cassyrane and oxidizing the resulting seco‐structure to the 1,2,5,1″‐tetradehydro derivative links the family of dienone musks with that of blackcurrant odorants, but the resulting target structures turned out to be potent orris odorants. (3E,5E)‐5‐(tert‐Butyl)octadeca‐3,5‐dien‐2‐one even possesses the lowest odor threshold in the whole ionone family (0.036 ng/l air), which could be rationalized by a superposition analysis on (?)‐cis‐γ‐irone. In the course of the synthesis of these high‐impact orris odorants, we discovered that, depending on the reaction conditions, the dehydration step of the intermediate 5‐hydroxyalk‐3‐yn‐2‐ones was accompanied by a carbenium‐ion rearrangement. Depending on the substitution pattern, these rearrangement products and their derivatives possessed interesting musky‐woody olfactory properties reminiscent of Cashmeran, demonstrating that the same structural elements can code for completely different odors, i.e., cassis, musk, orris, violet, and Cashmeran‐type, depending only on their spatial arrangement.     

New alicyclic musks: the fourth generation of musk odorants

Musk odorants are one of the most important classes of fragrances in perfumery because they impart sensuality to perfume-oil compositions. Among the three well-known classes of musks, a new and very exciting generation of musk odorants, the so-called alicyclic musks, was discovered recently, of which Helvetolide (2) and Romandolide (3) are the most popular representatives so far. To find new, structurally related alicyclic musks, we have synthesized a library of 114 unique alicyclic molecules with modified cyclohexyl moieties. The olfactory properties of all compounds were evaluated to identify the structural requirements to be met for a musk odorant.     

Construction of a Quantitative Three-dimensional Model for Odor Quality using Comparative Molecular Field Analysis (CoMFA)

A quantitative structure-activity relationship (QSAR) studyof odorants was performed taking an odor as an activity. Asan example, we took the ‘green odor of pyrazine derivatives’as an activity. Conformational analysis of the pyrazine derivativeswas performed, and conformers were selected using the longestside-length of a circumscribed box (LLCB) as a criterion. Comparativemolecular field analysis (CoMFA) was used to elucidate the three-dimensional(3D) structural features of the derivatives. As a result, itwas found that the steric and electrostatic features of thederivatives were correlated with human olfactory detection thresholdvalues. We constructed a quantitative 3D model using the graphicviews of CoMFA and partial structures of the derivatives. Theprediction of human olfactory detection threshold values ofother pyrazine derivatives with green odor was possible by usingthe 3D model. As another example, we took the ‘sweet odorof compounds with various structures’ as an activity.A quantitative 3D model for sweet odor was constructed in thesame manner. Analysing the structural features of odorants byCoMFA and constructing 3D models for several important odorqualities would help to (i) explain or predict human olfactorydetection threshold values of interesting odorants, (ii) designnew odorants by suggesting the steric and electrostatic requirements,and (iii) elucidate the mechanism of odorant-receptor interaction.Chem Senses 21: 201–210, 1996.     

Woody pretzels: spirocycles from vetiver to patchouli and Georgywood

This review, including new experimental results, is the summary of a talk at the RSC/SCI conference 'flavours & fragrances 2007' in London, Imperial College, 24-26 September, 2007. Though the third dimension of the receptor models of J. E. Amoore rarely was exceeding 4 A, the world of woody odorants such as (+)-cedrol (3; cedarwood), (-)-khusimone (4; vetiver), and (-)-patchoulol (5; patchouli) is anything but flat. Any tricyclic skeleton with a zero-bridge contains a spirocyclic ring system determining its 3D structure, so spirocycles (spira, Lat. pretzel) are the fastest access to the third dimension. In the vetiver family, a spirocyclic mimic 9 of (-)-khusimone (4) was first discovered by chance by Büchi in 1976, and also by chance, we obtained another system, 12, with a characteristic vetiver smell by tandem-Rupe-Nazarov reaction of alkyne diols. A 5-A distance between a quaternary C-atom and a carbonyl group (or alternative HB acceptor) with an alpha-methyl or methylene branching is proposed to be the key to their vetiver odor. Upon scale-up of one of these odorants, 24, we discovered a very powerful (0.067 ng/l) impurity with a most typical patchouli scent: the spirocyclic, sterically crowded hydroxy ketone 33--a most unusual structure for a patchouli odorant. Several spirocyclic hydroxy ketone analogs, also with inverted ring systems such as in 70 and 84, provided new insights into the structure-odor correlation of this family. A superposition analysis indicated the carbonyl function of the hydroxy ketone to overlay on the geminal dimethyl motive of (-)-patchoulol. And indeed, the corresponding hydroxy ketone of patchoulol, 59, synthesized in 13 steps from Cyclal C (63), also emanated a patchouli odor. Finally, the synthesis and olfactory properties of twelve rigid spirocyclic analogs, 95-97, 99-102, and 106-110, of Georgywood (91) are presented that highlight stereochemical requirements for woody odorants and raise doubts about an alpha-helical binding motive postulated by Hong and Corey.     

The search for innovative fragrant molecules

An overview of the recent research subjects in the perfumery field and, more precisely the synthesis of novel compounds for the hesperidic family of odorants, is presented. The new derivatives possess very different structures; nevertheless, they all share an aldehydic odor, but with diverse nuances, which make them all attractive for different purposes.     

Odorant-receptor interactions and odor percept: a chemical perspective

Receptor-ligand interaction models are generally based on a 'lock and key' concept. How far this holds true for olfactory receptors and odor molecules is currently uncertain. Here, we have investigated the response of a human olfactory receptor, OR1D2, to a broad array of odorants and found that there is no simple, direct correlation between a molecule's ability to activate this receptor and the odor impression elicited in the brain. In a parallel study on specific anosmia, we have found no evidence for odor-specific anosmia to either musk or amber, but rather to specific molecules within these categories. Cluster analysis confirmed that there is no simple correlation between molecular structure and impaired perception in either odor type. There are some differences in patterns of impairment between the two odor types and some evidence to suggest that subjects with specific anosmia to a given substance can identify its presence in a mixture. Taken together, our results show that simplistic 'lock and key' models of olfaction based on a concept of odor-quality-tuned receptors are inadequate, irrespective of the nature of the lock-key interaction. Receptor activation is only one step in a long chain of events leading from inhalation of odorants to perception of odor in the higher brain, and, therefore, although structure-odor correlations are useful tools for the design of novel odorants, caution should be exercised when extrapolating them to models of olfactory perception. Those seeking to understand the odorant-receptor interaction should use receptor activation rather than odor as input data.     

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors^1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.     

Odor Perception Phenotypes: Multiple, Specific Hyperosmias to Musks

Olfactory detection thresholds for 11 structurally diverse muskodorants and one non-musk odorant were obtained from 32 subjects.Hierarchical cluster analysis produced four groups of subjects.One group (n = 12) was uniformly sensitive to all musks; another(n = 16) was uniformly insensitive. Two groups of subjects containedotherwise insensitive individuals who were exceptionally sensitiveto cyclopentadecanone and musk xylol (n = 2) and to delta9-hexadecenolactoneand tonalid (n = 2) respectively. We propose that the lattertwo groups are odor perception phenotypes (MSHM1 and MSHM2)that consist of multiple, specific hyperosmias to musk odorants.Chem. Senses 21: 411– 416, 1996. ¹Present address: Synesthetics, Inc., Montclair, NJ 07043, USA     

A method for the calculation of odor character from molecular structure

The relationship between molecular structure and odor character is one of the most complex structure-activity problems in biology. Despite over a century of effort, it remains unsolved, and synthesis of new odorants still proceeds largely by trial and error. In previous work, I have argued that the reason for this failure lies in a mistaken assumption, namely that molecular shape determines odor character. Instead, I have taken up and extended an old idea (Dyson, 1938) according to which vertebrate olfactory receptors detect odorants by their molecular vibrations. I propose that the detection mechanism is inelastic electron tunnelling. If this is correct, there should be a correlation between the tunnelling vibrational spectra of odorants and their odor character. Here, using semi-empirical quantum chemistry methods and a simple calculation method for tunnelling mode intensities, I calculate the spectra of structurally diverse odorants belonging to various odor categories. With few exceptions, the calculated spectra of bitter almonds, musks, ambers, woods, sandalwoods and violets strongly correlate with odor character.     

Odor-active constituents in fresh pineapple (Ananas comosus [L.] Merr.) by quantitative and sensory evaluation

By application of aroma extract dilution analysis (AEDA) to an aroma distillate prepared from fresh pineapple using solvent-assisted flavor evaporation (SAFE), 29 odor-active compounds were detected in the flavor dilution (FD) factor range of 2 to 4,096. Quantitative measurements performed by stable isotope dilution assays (SIDA) and a calculation of odor activity values (OAVs) of 12 selected odorants revealed the following compounds as key odorants in fresh pineapple flavor: 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF; sweet, pineapple-like, caramel-like), ethyl 2-methylpropanoate (fruity), ethyl 2-methylbutanoate (fruity) followed by methyl 2-methylbutanoate (fruity, apple-like) and 1-(E,Z)-3,5-undecatriene (fresh, pineapple-like). A mixture of these 12 odorants in concentrations equal to those in the fresh pineapple resulted in an odor profile similar to that of the fresh juice. Furthermore, the results of omission tests using the model mixture showed that HDF and ethyl 2-methylbutanoate are character impact odorants in fresh pineapple.     

From musk to body odor: Decoding olfaction through genetic variation

The olfactory system combines input from multiple receptor types to represent odor information, but there are few explicit examples relating olfactory receptor (OR) activity patterns to odor perception. To uncover these relationships, we performed genome-wide scans on odor-perception phenotypes for ten odors in 1000 Han Chinese and validated results for six of these odors in an ethnically diverse population (n = 364). In both populations, consistent with previous studies, we replicated three previously reported associations (β-ionone/OR5A1, androstenone/OR7D4, cis-3-hexen-1-ol/OR2J3 LD-band), but not for odors containing aldehydes, suggesting that olfactory phenotype/genotype studies are robust across populations. Two novel associations between an OR and odor perception contribute to our understanding of olfactory coding. First, we found a SNP in OR51B2 that associated with trans-3-methyl-2-hexenoic acid, a key component of human underarm odor. Second, we found two linked SNPs associated with the musk Galaxolide in a novel musk receptor, OR4D6, which is also the first human OR shown to drive specific anosmia to a musk compound. We noticed that SNPs detected for odor intensity were enriched with amino acid substitutions, implying functional changes of odor receptors. Furthermore, we also found that the derived alleles of the SNPs tend to be associated with reduced odor intensity, supporting the hypothesis that the primate olfactory gene repertoire has degenerated over time. This study provides information about coding for human body odor, and gives us insight into broader mechanisms of olfactory coding, such as how differential OR activation can converge on a similar percept.     

Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe

A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain [1-7]. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants [8-11]. OSNs also exhibit odor-evoked responses with complex temporal dynamics [11], but the contribution of this activity to behavioral odor discrimination has received little attention [12]. Here, we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.     

Combinatorial receptor codes for odors

The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odors. Here we used a combination of calcium imaging and single-cell RT-PCR to identify odorant receptors (ORs) for odorants with related structures but varied odors. We found that one OR recognizes multiple odorants and that one odorant is recognized by multiple ORs, but that different odorants are recognized by different combinations of ORs. Thus, the olfactory system uses a combinatorial receptor coding scheme to encode odor identities. Our studies also indicate that slight alterations in an odorant, or a change in its concentration, can change its "code," potentially explaining how such changes can alter perceived odor quality.     

Olfactory discrimination ability for aromatic odorants as a function of oxygen moiety.

To assess the significance of the type of oxygen moiety on odor quality of aromatic compounds, I tested the ability of human subjects to distinguish between odorants sharing a benzene ring and the same total number of carbon atoms but differing in their functional groups. Phenyl ethanol, phenyl acetaldehyde, phenyl methyl ketone, methyl benzoate and phenyl acetic acid, were employed. In a forced-choice triangular test procedure 20 subjects were repeatedly presented with all possible binary combinations of the five odorants, and asked to identify the bottle containing the odd stimulus. I found (i) that as a group, the subjects performed significantly above chance level in six of the tasks whereas they failed to do so with the four other tasks; (ii) marked interindividual differences in discrimination performance, ranging from subjects who were able to significantly distinguish between all 10 odor pairs to subjects who failed to do so with the majority of the tasks; and (iii) that odor pairs that involved methyl benzoate or phenyl methyl ketone were significantly easier to discriminate than those that involved phenyl acetaldehyde or phenyl ethanol, and thus there was a clear dependence of discriminability on type of functional group. Additional tests of the degree of trigeminality of the five aromatic substances indicated that the discriminability of the odor pairs is indeed due to differences in odor quality. A comparison of the present results with those of an earlier study that employed aliphatic odorants suggests that functional oxygen-containing groups may generally be an important determinant of the interaction between the stimulus molecule and the olfactory receptor, and thus may generally be a molecular property affecting odor quality in a substance class-specific manner. The poorer discriminatory performance of the subjects with aromatic odorants compared with corresponding aliphatic substances suggests that the structure of the alkyl rest attached to a functional group may also play a crucial role for recognition of ligands at the olfactory receptor and thus for odor quality.     

An odorant derivative as an antagonist for an olfactory receptor

Different odorants are recognized by different combinations of G protein-coupled olfactory receptors, and thereby, odor identity is determined by a combinatorial receptor code for each odorant. We recently demonstrated that odorants appeared to compete for receptor sites to act as an agonist or an antagonist. Therefore, in natural circumstances where we always perceive a mixture of various odorants, olfactory receptor antagonism between odorants may result in a receptor code for the mixture that cannot be predicted from the codes for its individual components. Here we show that stored isoeugenol has an antagonistic effect on a mouse olfactory receptor, mOR-EG. However, freshly purified isoeugenol did not have an inhibitory effect. Instead, an isoeugenol derivative produced during storage turned out to be a potent competitive antagonist of mOR-EG. Structural analysis revealed that this derivative is an oxidatively dimerized isoeugenol that naturally occurs by oxidative reaction. The current study indicates that as odorants age, they decompose or react with other odorants, which in turn affects responsiveness of an olfactory receptor(s).     

Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein

Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSH(D118A), produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSH(D118A) reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants.     

Ontogeny of odor liking during childhood and its relation to language development

One important aspect of odor hedonics is its plasticity during human development. The present study set out to probe the modulators of such olfactory change during that period by testing the hypothesis that language and semantic representations of objects are strong organizers of odor liking. To this end, 15 three-year-old children were tested in a longitudinal study. Participants were exposed to exactly the same 12 odorants once a year over a 3-year period. At each experimental session, they were asked to answer 2 questions: 1) "Do you like or dislike this odor?" and 2) "Can you tell me what it is?" The level of language production was assessed on a standardized test. The 3-year-old children were found to categorize the same number of odorants as liked and as disliked. The follow-up study, in contrast, showed that at 5 years of age they categorized more of these odors as liked and that the shift was significant only in the children with higher language production skills. Taken as a whole, these findings suggest that the 3- to 5-year age range, when children begin to master language, is a turning point in the construction of olfactory hedonic categories during childhood.     

Structure-activity studies on odor molecules using molecular connectivity

Several classes of odorants have been examined using a newly developed structural analysis known as molecular connectivity. Within each class significant correlations have been found between odor similarities to a reference standard and connectivity terms. In each case some interpretation is possible as to certain structure-activity relationships imparting odor.     

Olfactory discrimination ability and odor structure-activity relationships in honeybees.

Using the training procedure introduced by von Frisch in 1919, we tested the ability of free-flying honeybees to discriminate a conditioning odor from an array of 44 simultaneously presented substances. The stimuli included homologous series of aliphatic alcohols, aldehydes and ketones, isomeric forms of some of these substances, as well as several terpenes and odor mixtures, and thus comprised stimuli of varying degrees of structural similarity to any conditioning odor. We found (i) that the honeybees significantly distinguished between 97.0% of the 1848 odor pairs tested, thus showing an excellent discrimination performance when tested in a free-flying situation with an array of structurally related substances; (ii) a significant negative correlation between discrimination performance and structural similarity of odorants in terms of differences in carbon chain length with all aliphatic substance classes tested; (iii) that both the position and type of a functional group also affected discriminability of odorants in a substance class-specific manner; and (iv) striking similarities in odor structure-activity relationships between honeybees and human and nonhuman primates tested previously on a subset of substances employed here. Our findings demonstrate that the similarities found in the structural organization of the olfactory systems of insects and vertebrates are paralleled by striking similarities in relative discrimination abilities. This strongly suggests that similar mechanisms of odor coding and discrimination may underlie olfaction in vertebrates and insects.